Nonapeptides and method of manufacture



- protective grouping or groupings present in a compound ICC.

' ,256,267 The compounds IV above may be obtained by con- NONAPEPTIDES AND METH D densing a tetrapeptide of the general Formula II,

United States Patent 0 MANUFACTURE HN=C Roger Boissonnas, Bottmingen, Basel Land, Switzerland,

assignor to Sandoz Ltd. (also known as Sandoz A.G.), NH Basel, Switzerland, a Swiss firm E No Drawing. Filed May 29, 1961, Ser. No; 113,129 7 Claims priority, application Switzerland, June 2, 1960, H2 CH1 2 6,334/60 (111, ed, on, 0%, CHz 8 Claims. (Cl. 260-1125) The present invention relates to a hitherto unsyn- 1O thesized polypeptide and to a process for its manufacture. 1 OH The nonapeptide having the Formula I, g y g I 1 CH2 CH1 C0115 OH CH1 C5115 I I CH (I111; (1H1 CH (IE (111 (3112 on, CIH1 N-(|3H N (|JH NH('JH2 NH(13H NH(|JH N(IJH NH-CH NH 0 0 0 0 o o (5 II II H II II II I] O O O O O O O 0 NH: NH: N H! has been synthesized now for the first time. It has been in which found that the nonapeptide I is identical with the naturally occurring bradykinin of which the structural in and formula has hitherto been unknown. Bradykinin was I first discovered by Rocha e Silva, M., Beraldo, W. T., has the above slgm'ficance and Rosenield, G Amen L physiol', 156 261 (1949) either in the form of the free acid or a reactive derivative twelve years before the synthesis of the nonapeptide I. thereof Wlth a Penmpepude of the formula Since the discovery of bradykinin, various attempts were made to elucidate its structure but without success; until NH immediately before its present synthesis by the inventor of the present invention, bradykinin was thought to be an oc- 2 R represents a protective grouping for an amino group- O H OH CH; C H CH3 tapeptide [see Elliott, D. F., Horton, E. W., and Lewis,

G. P., J. PhysioL, 150, 6 P (1960)]. It is stressed that 011, OH; on, on, 011, CH;

the nonapeptide I was synthesized by the present inventor NHABH NH CH before the structure of bradykinin was known and that the I identity of bradykinin with the nonapeptide I was estab- 40 H H H lished after the last mentioned compounds synthesis [see '0 0 0 0 0 III Nature, vol. 188, No. 4755, p. 998, December 17, 1960]. in which R" and R' have the above significance. Suit- Thus, the present invention provides the nonapeptide of able protective groupings for amino groupings, are the the Formula I above, and nonapeptides having the general carbobenzoxy, tolylsulfonyl, the carbo-tertbut-oxy, the Formula IV, phthalyl, the formyl and the p-nitro-carbobenzoxy groups.

R represents a hydrogen atom or a protective grouping are the eafhobehZoXy, tolylshlfohyl and the hitl'o p for an amino group, Suitable protective groupings for the carhoxyl groupings R' and. R" each represent a hydrogen atom or a proare the y the P- y the y the y tective grouping for a guanido grouping, and and the teft-hhtyl groups. R' represents a hydrogen atom or a protective group- The nonapeptide I strongly lowers the blood pressure ing for a carboxylic acid grouping with the proviso that and has a high Vascular activity which y be made use at least one of R, R, R" and R' must be a protective of in treatment of peripheral circulation troubles and grouping hypertony. J

The present invention also includes pharmaceutical one method of Predhelhgv e0IT1P0uhd I above t ey e compositions containing, in addition to an inert carrier, effected as touows: Nc3Tb0behzexy-hltre-L-afglhlhe I the nonapeptide I above. condensed with the methyl ester of L-proline and the re- The present invention also provides a process for the W e: P Pt ester is eapohified e Y the p n production of the nonapeptide 1, characterized i th t th mg acid. The'last mentloned material 1s condensed with the tertiary butyl ester of L-prolyl-glycine and the result- IV is or are split off. ing tetrapeptide ester is saponified by treatment with hyminutes.

drogen chloride to give the free acid of the Formula II above. Furthermore, the p-nitrobenzyl ester of nitro-L- arginine is condensed with N-carbobenZoxy-L-phenylalanine, the carbobenzoxy group is selectively split off with hydrogen bromide in glacial acetic acid, the resulting dipeptide is condensed with N-carbobenzoxy-L-proline, the carbobenzoxy group is split off in similar manner and the resulting tripeptide is condensed with N-carbobenzoxy- L-phenylalanine-L-seryl aZide. The last mentioned material is obtained by condensation of N-carbobenzoxy-L- phenylalanine with the methyl ester of L-serine and conversion of the resulting dipeptide ester into the azide via the hydrazide. The resulting pentapeptide has the carbobenzoxy group split off with hydrogen bromide in trifiuoro acetic acid giving the pentapeptide III which is condensed with the tetrapeptide II to give the corresponding nonapeptide having the general Formula IV above, from which the nonapeptide I is obtained by hydrogenolysis of the protective groupings.

In the immediately preceding specific method of producing the compound I it is possible to split oif the protective groupings R and R of the pentapeptide before the condensation with the tetra-peptide. It is likewise possible to use for the condensation a tetrapeptide in which R represents a hydrogen atom.

Various other condensation procedures using peptides containing the required number of amino acid groups, or even a peptide with an amino acid, may be substituted for the above described specific method of producing the nonapeptide I as is well known in the field of polypeptide chemistry, and such variations are also within the scope of invention.

In the process of the invention the splitting oil of the protective groupings may be effected, inter alia, by hydrogenolysis, for example with an alkali metal in liquid ammonia, or by catalytic hydrogenation.

The following examples illustrate the invention Without, however, limiting it. Temperatures are expressed in degree centigrade.

EXAMPLE 1 (a) N-carbobenzoxy-nitro-L-arginyl-L-proline 35.5 g. of N-carbobenzoxy-nitro-L-arginine, 12.9 g. of L-proline methyl ester and 24.8 g. of dicyclohexylcarbodiimide are dissolved at in 250 cc. of dimethyl formamide and 100 cc. of acetonitrile, the solution being allowed to stand overnight at and then boiled down in a vacuum. The residue is triturated in petroleum ether, dissolved in ethyl acetate, and the filtrate is washed with dilute hydrochloric acid and dilute ammonia. The ethyl acetate is evaporated in a vacuum, the residue dissolved in 300 cc. of methanol and 300 cc. of a 1 N-solution of caustic soda, and the solution kept for 1 hour. 500 cc. of water are then added, followed by filtration, acidification with dilute hydrochloric acid and extraction with ethyl acetate. After evaporation of the ethyl acetate the residue crystallises when methanol is added. 23.0 g. of N-carbobenzoxy-nitro-L-arginyl-L-proline are obtained, M.P. 119; [a] =47 (0. 2 in glacial acetic acid).

(b) N-carbobenzoxy-nitro-L-arginyl-L-prolyl-L- prolyl glycine 9.6 cc. of chloro fonmic ethyl ester are added at 10 to a solution of 24.9 g. of N-canboibenzoxy-L-proline and 14.0 cc. of triethylamine in 300 cc. of chloroform, 11.3 g. of glycine-tert.-butyl ester being added thereto after 10 The mixture is leit 'for two hours at room temperature, washed with dilute ammonia and dilute phosphoric acid, evaporated in a vacuum, dissolved in methanol, and the carbobenzoxy group is then split off by catalytic hydrogenation. The solution is boiled down, the residue dissolved in 250 cc. of dimethyl formamide and 100 cc. of acetonitrile, and 40.0 g. of N-carbobenzoxynitro-L-arginyl-L-p-roline together with 22.0 g. of dicyclohexyloarbodiimide are then added. The mixture is allowed to stand overnight at 20 and evaporated in a a vacuum. The residue is triturated in petroleum ether, dissolved in chloroform, washed with dilute phosphoric acid and dilute ammonia and dried on sodium sulphate. Dry hydrogen chloride is then passed through the solution for 4 hours at 35. After boil-ing down in a vacuum and adding ethyl ether, 30.2 g. of 'N canbobenzoxy-nitro-L- arginyl-bprolyl-Lprolyl glycine are obtained. M.P. 115v under decomposition. Eq. weight 605. Found 602. (c) N-carbobenzoxy-L-phenylalanyl-L-seryl-azide 30.0 g. of \N-carbobenzoxyL4p=henylalanine, 15 g. of L-serine methyl ester hydrochloride, 14 cc. of triethylamine and 24.8 g. of dicyclohexylcarhodiimide are dissolved in 1500 cc. of aoetonit-rile at 10. The solution is allowed to stand overnight at 20, filtered, boiled down in a vacuum, and the residue is triturated in petroleum ether, .re-dissolved in ethyl acetate, washed With dilute hydrochloric acid and dilute ammonia, dried on sodium sulphate and precipitated with petroleum ether. The precipitate is dissolved in 300 cc. of methanol, and 12 cc. of hydrazine hydrate are added. The solution is allowed to stand overnight at 20, then filtered and washed with menthanol and ether. 28.2 g. of hydrazide of M.'P. 193 are obtained, which are dissolved in cc. of glacial acetic acid and 100cc. of a 2 N-hydrochloric acid. After the solution has been cooled :to 5, 15 cc. of a 5 N-solu- 'tion of sodium nitrite and, 10 minutes later, 450 cc. of ice cold water are added. The precipitate is filtered off, washed with water and rapidly dried in a vacuum. 26.1 g. of the az-ide are obtained and immediately used. (d) L-phenylalanyl-L-seryl-L-prolyl-L-phenylalanyl-nitro-L-argiitine-pmitro-benzyl ester 106 g. of N-carbobenzoxy-nitro-larginine, 63 cc. of triethylam-ine and 77 g. of p-nitro-lbenzylchloride are dissolved in 450 cc. of dimethyl fiormamide and kept at 80 overnight. The solution is then boiled down in a vacuum, the residue dissolved in ethyl acetate, washed with dilute hydrochloric acid and dilute ammonia, dried on sodium sulphate and again boiled down in a vacuum. The crystallised residue is dissolved in 1100 cc. of a 2.2 .N-solution of hydrogen bromide in glacial acetic acid and left for exactly 20 minutes at 20. The solution is evaporated in a vacuum, ethyl ether is added, and the crystallised residue is dissolved in 300 cc. of dimethyl iformamide and 35 cc. of triethylamine. This solution is poured into a \freshly prepared mixture obtained by adding 14.5 cc. of chlorofonmi-c ethyl ester at 10 to a solution of 45 g. of N-canbobenzoxy-L-phenyl alanine and 21 cc. of trie thylamine in 750 cc. of tetrahydrofuran. The mixture is allowed to stand overnight at 20, and then evaporated in a vacuum. The residue is dissolved in 500 cc. of ethyl acetate, washed with dilute hydrochloric acid and dilute ammonia and dried on sodium sulphate. A fiter the solution has been allowed to stand for several hours at 5, 56 g. of'N-canbobenzoxy-L phenylalanyl-nitro-L-arginine p-nitro-benzyl ester (MJP. 174) are filtered off and dissolved in 550 cc. of a 2.2 N- solution of hydrogen bromide in glacial acetic acid. The solution is left for 20 minutes at 20 and then boiled down in a vacuum, 3000 cc. of ethyl ether being added and the crystallised residue M.P. 175) dissolved in 150 cc. on. chloroform and '19 cc. of triethylamine. The

- solution is poured into a freshly prepared mixture obtained by adding 9.0 cc. of chlorotorm-ic ethyl ester at 10 to a solution of 23 g. of N-canbobenzoxy-Lproline and 13 cc. of triethylamine in 150 cc. of chloroform. After having allowed this mixture to stand at 20 for three hours it is'evaporated in a vacuum, the residue is dissolved in ethyl acetate, Washed with dilute hydrochloric acid and dilute ammonia, dried on sodium sulphate, again boiled down in a vacuum, and ethyl ester is added to the residue. 49 g. of the tripeptide ester (M.-P. are obtained and dissolved in 450 cc. of a 2.2 N-solution of hydrogen bromide in glacial acetic acid. After having been left at 20 for 20 minutes the solution is again boiled down in a vacuum. Ethyl ether is added to the residue which is dissolved in a mixture of 450 cc. of ethyl acetate, 225 cc. of acetoni'trile and 150 cc. of an aqueous saturated solution of potassium carbonate. The organic phase is separated off, dried on sodium sulphate and 1 evaporated in a vacuum. The residue is dissolved in 300 cc. of dimethyl for-malm-ide and 26.1 g. of N-carbobenzoxy-L-phenyla-lanyhL-seryhazide are added. This is allowed to stand at 0 for two days before it is evaporated in a vacuum, ethyl ether being added to the residue and the product recrystallised from isopropanol. The pentapeptide is dissolved in 500cc. of trifluoroacetic acid, the solution being saturated with hydrogen bromide at 0 and then boiled down in a vacuum after having stood at 0 tfor one hour. Ethyl ether isadded to the residue which is dissolved in a mixture of 300 cc. of dimethyl form- =amide and 300 cc. of ethyl acetate and shaken out with 50 cc. of an aqueous saturated solution of potassium carbonate. The organic phase is dried on sodium sulphate and evaporated in a vacuum.

The addition of ethyl ether yields 37 g. of L-phenylalanyl L seryl L prolyl L phenylalanyl nitro L- arginine-p nitro-benzyl ester.

(e) L arginyl L prolyl L prolyl glycyl L phenylalanyl L seryl L prolyl L phenylalanyl L- arginine.

30 g. of N-carbobenzoxy-nitro-L-arginyl-L-prolyl-L- prolyl-glycine, 37 g. of L-phenylalanyl-L-seryl-L-prolyl- L-phenylalanyl-nitro-L-arginine-p-nitro-benzyl ester and 12 g. of dicyclohexylcarbo-diimide are dissolved at in 300 cc. of dirnethylformamide and 100 cc. of acetonitrile, the solution being allowed to stand overnight at 20", then filtered and evaporated in a vacuum, the residue being suspended in 1000 cc. of boiling ethyl acetate. When this has cooled 55 g. of protected nonapeptide are obtained, which are dissolved in a mixture of 750 cc. of glacial acetic acid, 100 cc. of a 2 N-hydrochloric acid and 650 cc. of water. For 24 hours this is hydrogenated at atmospheric pressure in the presence of a platinum catalyst as described by Van Orden and Smith [1. Biol. Chemistry 208, 751 (1954)]. After filtration and evaporation in a vacuum, the residue is crystallised from isopropanol/water/ethyl ether. 34 g. of the dihydrochloride of L arginyl L prolyl L prolyl glycyl L- phenylalanyl L seryl L prolyl L phenylalanyl L- arginine are obtained. The nonapeptide has an isoelectric point of 10.5. When submitted to acid hydrolysis it yields arginine, proline, glycine, phenylalanine and serine in the proportions of 2:3:1:2:1.

EXAMPLE 2 30.2 g. of N-carbobenzoxy-nitro-L-arginyl-L-prolyl-L- prolyl glycine and 7.0 cc. of .triethylamine are dissolved in 500 cc. of dioxane at 10 with 4.8 cc. of chloroformic acid ethyl ester, and after 10 minutes 32.6 g. of L phenylalanyl L seryl L prolyl L phenylalanyl- L-arginine (prepared for instance by hydrogenatin-g N- carbobenzoxy L phenylalanyl L seryl L prolyl- L phenylalanyl nitro L arginine p nitro benzyl ester) in 200 cc. of water and 7 cc. of triethylamine are added. The solution is stirred for 5 hours at 20 and I proline-methylester-chlorohydrate and 24.8 g. of dicyclohexylcarbo-diimide are dissolved at l0 in 250 cc. of dimethyl formamide and cc. of acetonitrile, the solution being allowed to stand overnight at 20 and then boiled down in a vacuum. The residue is triturated in petroleum ether, dissolved in 300 cc. of methanol and 300 cc. of a 1 N-solution of caustic soda and again left for 1 hour at the end of which 500 cc. of water are added, the solution being filtered, neutralised with dilute hydrochloric acid, again filtered, washed with water and dried.

28 g. of N-carbobenzoxy-L-arginine-L-proline are obtained and dissolved in 250 cc. of dimethyl formam-ide and 100 cc. of acetonitrile. At 10, 18 g. of dicyclohexylcarbo-diimide, 16.5 g. of L-prolyl-glycine-tert.-butyl ester and 12 g. of toluene-sulphonic acid are added and the mixture is allowed to stand for 16 hours at room temperature before being precipitated with ethyl ether, dissolving the precipitate in 200 cc. of glacial acetic acid, passing dry hydrogen chloride through the solution for 2 hours, evaporating, adding 100 cc. of a 1 N-solution of caustic soda and 100 cc. of methanol, neutralising with hydrochloric acid, filtering and washing with water and isopropanol. 32.5 g. of N-carbobenzoxy-L-arginyl-L- prolyl-L-prolyl-glycine (eq. weight 560; found 557) dissolved in 200 cc. of dimethyl formamide and 50 cc. of acetonitrile in the presence of 9.9 g. of toluene sulphonic acid, 20 g. of p-nitro-phenol and 20, g. of dicyclohexylcarbo-diimide are added and the mixture is allowed to stand at room temperature overnight. 32.6 g. of L-phenylalanyl L seryl L prolyl L phenylalanyl L- arginine (prepared, for instance, by hydrogenating N-carbobenzoxy L phenylalanyl L seryl L prolyl L- phenylnitro-L-arginine-p-nitro-benzyl ester), 100 cc. of water and 7 cc. of triethylamine are added. After having stood at room temperature for 16 hours the solution is boiled down in a vacuum and the residue recrystallised from isopropanol and water. 41 g. of N-carbobenzoxy- L arginyl L prolyl L prolyl glycyl L phenylalanyl L seryl L prolyl L phenylalanyl L arginine are obtained which are dissolved in 500-cc. of glacial acetic acid and 500 cc. of a 1 N-hydrochloric acid and hydrogenated for one hour at atmospheric pressure in the presence of a platinum catalyst. The further procedure is analogous to that described in Example 1, section (e).

' EXAMPLE 4 The procedure is as described in Example 3 but for detaching thecarbobenzoxy group, the nonapeptide is dissolved in 4 litres of liquid ammonia and treated with metallic sodium whilst being stirred until the solution turns blue. After evaporating the ammonia the precedure continues as described in Example 1, section (e).

I claim:

1. A process for the production of the nonapeptide 1 2 8 comprising splitting off the protective groupings R, R, from the group consisting of hydrogen and a protective R, R' present in a compound having the formula IV grouping for a carboxylic acid grouping consisting of /Cgz /Cgz 05115 (RE /Cg2 (Isl CH2 (IJHs (EH2 CH2 CH2 (i lHz (EH2 (3H2 (1H1 N--( H NCH NH-(fHz NHC|1H/NH(|1H/N({3&NH(|3H NH I o o 0 o o o C II II II H H II II 0 O o 0 0 0 0 w r r (13ft? Hc HzC Hz-C HPNH-(IJ (|3NHC H2C H2O 112-0 11-? o ITIH IIIH #IH R R R" IV wherein R is selected from the group consisting of hydrothe benzyl-, the p-nitrooenzyl-, the methyl-, the ethylgen and a protective grouping for the amino group, said and the tertiary-butyl groups, at least one of R, R, R amino protective grouping being a radical selected from and R being a protective grouping which is split off the class consisting of the carbobenzoxy-, the tolueneby known methods in peptide chemistry. sulfonyl-, the carbo-tertiary-butoxy, the phthalyl-, the 3. The compound corresponding to the Formula IV /Cgz /CI\'I2 (ll rn on /Cg C5H5 (fliz CIJI'IZ CH2 CH2 CH2 (fl-I2 (3E2 (311: (IJH N IJ3N CL NH(E@NH(E$NHoILNoH NH-(IJH NH I o o o o 0 o l II II II II II II H o 0 0 0 0 0 o l r t (5-? 11-0 Hz-C Hz-C H2NH(E |3-NH-C I-Iz-C H-C H2-O H(') 0 I| IH 1TH ITIH R R R IV formyl-and thep-nitro-carbobenzoxy groups, R and R" are each selected from the group consisting of hydrogen 40 wherein R is selected from the group consisting of carand a protective grouping for a guanido-grouping selected bobenzoxy, toluenesulfonyl, the carbo-tertiary-butoxy, from the group consisting of the carbobenzoxy-, the the phthalyl, the formyl and the p-nitro-carbobenzoxytoluenesulfonyland the nitro groups and R is selected groups, R and R are each selected from the group confrom the group consisting of hydrogen and a protective sisting of hydrogen, the carbobenzoxy, the toluenesulfonyl grouping for a carboxylic acid grouping selected from the and the nitro groups, and R is selected from the groups group consisting of the benzyl-, the p-nitrobenzyl-, the consisting of hydrogen, the benzyl, the p-nitrobenzyl, the methyl-, the ethyland the tertiary-butyl groups, at least methyl, the ethyl and the tertiary butyl-group.

one of R, R, R", and R' being a protective grouping 4, N carbobenzoxy -L arginyl L prolyl L-prolylwhich is split ofi by saponification. glycyl L phenylalanyl L seryl L prolyl L-phenyl- 2. The compound corresponding to the Formula IV alanyl-L-arginin.

/0 H2 /Cr Clam ()H /CEZ (3 1-1 CH2 (IJHZ C|H2 (IJHZ CH3 (IJHQ CIHZ $11: C|Hz 1 TCH N-CH NH-C H2 NHCH NH-OH NGH NH-OH NH l/ l/ l l C 0 C o C II II ll II H H H 0 0 0 0 1 0 0 0 F W F CC HO Hz-CI-IzCHzNH (J (|JNHG Hi -o H -G H -C 11-41f NH ITIH 17TH I t R R IV wherein R is selected from the group consisting of hydrogen and a protective grouping for the amino group, 1 plinylalanyl L Beryl L prolyl L phenyl' said amino protective grouping being a radical selected a any from the class consisting of the carbobenzoXy-, the Ncarb0benzoxy'L'atgmyl'L'prolyl'L'prolyl'813/0111- toluene sulfonyl-, the carbo tertiary butoXy-, the 7 N carbobenzoxy nitlo L arginyl L P 3 phthalyl-, the formyland the p-nitro-carbobenzoxy piolyl-glycin.

groups, R and R are each selected from the group con 8. L phenylalanyl L L seryl L prolyl L phenylsisting hydrogen a Protective p for a a1anyl-nitro-L arginin-p-nitro-benzylester.

guanido grouping consisting of the carbobenzoxy-, the

toluenesulfonyland the nitro groups and R is selected (Rgfe ences on fgfl i Page) 9 I 10 References Cited by the Examiner Kpnzett: Nature, vol. 188, p 998 (1960). I 611 3.1.1 Amer. PhYSlOL, V01. pp. 2,793,204 5/1957 Arens. 2,854,443 9/1958 Boissonnas et a1. 5 LEWIS GOTIS primary Examiner 3,040,017 6/1962 Schwyzer 260112.5 3,131,174 4/1964 Schwyzer 260--112.5 LEON ZITVE Examiner- OTHER REFERENCES DENNIS P. CLARKE, PERRY STITH, Fuson: Advances in Protein Chemistry, vol. 5, p. 21-33 10 Assistant Examiners.

and 62-64 (1949). 

1. A PROCESS FOR THE PRODUCTION THE NONAPEPTIDE I 